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Interactions between heterotrophic bacteria and cyanobacteria regulate the structure and function of aquatic ecosystems and are thus crucial for the prediction and management of cyanobacterial blooms in relation to water security. Currently, abundant bacterial species are of primary concern, while the role of more diverse and copious rare species remains largely unknown. Using a dilution-to-extinction approach, rare bacterial species from reservoir water were co-cultured with the bloom-forming cyanobacterium Raphidiopsis raciborskii in the lab to explore their interactions by using Phyto-PAM and 16S rRNA gene high-throughput sequencing. We found that a ≤1000-fold bacterial dilution led to bacteria control of the growth and photosynthesis of R. raciborskii. Moreover, the bacterial community compositions in the low-dilution groups were clearly diverged from the high-dilution groups. Importantly, rare species changed dramatically in the low-dilution groups, resulting in lower phylogenetic diversity and narrower niche width. The network complexity and compositional stability of bacterial communities decreased in the low-dilution groups. Collectively, our results suggest that rare bacterial species inhibit R. raciborskii growth and photosynthesis through microbial interactions mediated by species coexistence and interaction mechanisms. Our study provides new knowledge of the ecological role of rare bacteria and offers new perspectives for understanding the outbreak and regression of R. raciborskii blooms.

In freshwater habitats, invasive species and the increase of cyanobacterial blooms have been identified as a major cause of biodiversity loss. The invasive cyanobacteria Raphidiopsis raciborskii a toxin-producing and bloom-forming species affecting local biodiversity and ecosystem services is currently expanding its range across Europe. We used species distribution models (SDMs) and regional bioclimatic environmental variables, such as temperature and precipitation, to identify suitable areas for the colonization and survival of R. raciborskii, with special focus on the geographic extent of potential habitats in Northern Europe. SDMs predictions uncovered areas of high occurrence probability of R. raciborskii in locations where it has not been recorded yet, e.g. some areas in Central and Northern Europe. In the southeastern part of Sweden, areas of suitable climate for R. raciborskii corresponded with lakes of high concentrations of total phosphorus, increasing the risk of the species to thrive. To our knowledge, this is the first attempt to predict areas at high risk of R. raciborskii colonization in Europe. The results from this study suggest several areas across Europe that would need monitoring programs to determine if the species is present or not, to be able to prevent its potential colonization and population growth. Regarding an undesirable microorganism like R. raciborskii, authorities may need to start information campaigns to avoid or minimize the spread.

Cyanobacterial blooms are intensifying global ecological hazards. The fine structure and dynamics of bloom community are critical to understanding bloom development but little understood. Here, the questions whether dominant bloomers have high diversity and whether dominant OTUs (operational taxonomical units) compete with one another were addressed. 16S rRNA gene amplicons from an annual bloom at five locations in Harsha Lake (Ohio, USA) showed cyanobacteria were the dominant phylum, and co-existing major bacterial phyla included Proteobacteria, Bacteroidetes, Actinoacteria, and Verrucomicrobia. On the genus level, the initial dominance by Dolichospermum in June yielded to Planktothrix in July, which were replaced by Microcystis and Cylindrospermopsis in August throughout the bloom. Based on the number of verified unique OTUs (a within-genus biodiversity metric), dominant genera tended to have high within-genus diversity. For example, Dolichospermum had 57 unique OTUs, Planktothrix had 36, Microcystis had 12, and Cylindrospermopsis had 4 unique OTUs. Interestingly, these different OTUs showed different dynamics and association with other OTUs. First, no between-OTU competitions were observed during the bloom cycle, and dominant OTUs were abundant throughout the bloom. Such biodiversity of OTUs and their dynamics were verified in Microcystis aeruginosa with two microcystin synthetase genes (mcyA and mcyG): the relative abundance of both genes varied during the bloom based on quantitative PCR. Two Dolichospermum circinale OTUs and one P. rubescens OTU were most abundant and persistently present throughout the entire bloom. Second, these OTUs differed in the OTUs they were associated with. Third, these OTUs tended to have different levels of association with the environmental factors, even they belonged to the same genera. These findings suggest the structure and dynamics of a cyanobacterial bloom community is complex, with only few OTUs dominating the bloom. Thus, high-resolution molecular characterization will be necessary to understand bloom development.

The concentration of coloured terrestrial dissolved organic matter (tDOM) from vegetation appears to be increasing in lakes in some regions of the world, leading to the term brownification. The light attenuating effect of coloured tDOM on phytoplankton growth has been a major focus of attention, but the phytotoxic effects of tDOM, particularly on cyanobacterial blooms, are less well understood. This mesocosm study tested whether coloured tDOM, leached from the leaves of a Eucalyptus tree species, inhibited a naturally occurring bloom of the toxic cyanobacterium, Raphidiopsis raciborskii, in a reservoir over a 10 day period. The study found that tDOM leachate, measured as dissolved organic carbon (DOC), inhibited photosynthesis and growth of both R. raciborskii, as well as species present at lower densities, i.e. other cyanobacteria and diatoms. However, the effect was greater at higher tDOM input loads. The photosynthetic yield (Fv/Fm) of cyanobacteria decreased rapidly in treatments with 5.9 and 25 mg L

Water bloom-forming cyanobacteria have a severe impact on freshwater quality. Although some cyanobacterial toxins such as microcystins have been studied extensively, other toxins like anatoxin-a (ATX) and their structural analogs - as well as cyanobacterial taxa producing these toxins remain to be explored in detail. The present study investigated levels of ATX, CYN and their homologs along with the occurrence of anaC and cyrJ genes in water blooms in 16 sites in the Czech Republic that were pre-selected concerning the presence of potential toxin producers. Besides, we also studied toxins and genes in a series of strains available in our laboratories. ATX and its congener HATX were detected in 5 natural biomass samples from the Czech Republic (maximum concentration 2.8 micrograms per gram d.w.). Interestingly, the anaC gene coding for ATX production was not detected in any of these toxin-positive biomass samples. The concentrations of ATX congeners in cyanobacterial laboratory strains were about 10-times higher than those of the original ATX, which calls for further research addressing levels and hazards of ATX analogs. Regarding the CYN and 7-deoxyCYN (other CYN congeners were not analyzed in this study) - these toxins were identified in a single small pond in the Czech Republic at concentrations 4.3 and 2.7 micrograms per gram of biomass d.w., respectively (corresponded to dissolved concentrations higher than 1 microgram per liter). The CYN-positive sample was dominated by CYN-producing taxa Raphidiopsis (basionym Cylindrospermopsis) and Cuspidothrix. We also confirmed the presence of a specific cyrJ gene in this natural bloom sample. To our knowledge, this is the first study pointing to Raphidiopsis (Cylindrospermopsis) and Cuspidothrix as producers of CYN in Europe. This observation calls for further research because of their increasing occurrence in (Central) Europe along with the global change. The present study demonstrates the importance of using combined (taxonomical, analytical, and molecular) approaches in the assessment of hazardous cyanobacteria and their toxins in freshwaters.

The extent and frequency of harmful cyanobacterial blooms are increasing, owing to the climate change caused by global warming, and some harmful filamentous cyanobacteria that were first reported in the tropics are spreading to temperate regions, such as North America, Europe, and Northeast Asia. Although these exotic invasive cyanobacteria have a high toxigenic potential, they are not targeted in management plans in many countries. This study analyzed the occurrence of and potential toxin and off-flavor secondary metabolite production by invasive nostocalean cyanobacteria in the Nakdong River in Korea, which is a temperate region. The occurrence of four species belonging to three genera of cyanobacteria was confirmed in the Nakdong River. The quantities of cyanobacteria in the Nakdong River were mostly low, fewer than 1,000 cells mL-1. Twenty-four strains belonging to four species in three genera of cyanobacteria were isolated from the Nakdong River. Analysis revealed no off-flavor secondary metabolite production by any of the isolates, and those belonging to Cylindrospermopsis raciborskii, Sphaerospermopsis aphanizomenoides, and S. reniformis were identified as nontoxic strains. However, anatoxin-a production was observed in two of the eleven isolates of Cuspidothrix issatschenkoi. Given the sites and the timing of its occurrence, C. issatschenkoi had the highest potential for toxin production among the invasive nostocalean cyanobacteria appearing in the Nakdong River.

Raphidiopsis raciborskii is a freshwater, potentially toxigenic cyanobacterium, originally described as a tropical species that is spreading to northern regions over several decades. The ability of R. raciborskii to produce cyanotoxins – in particular the alkaloid cylindrospermopsin (CYN), which is toxic to humans and animals – is of serious concern. The first appearance of R. raciborskii in Russia was noted in Lake Nero in the summer of 2010. This is the northernmost (57°N) recorded case of the simultaneous presence of R. raciborskii and detection of CYN. In this study, the data from long-term monitoring of the R. raciborskii population, temperature and light conditions in Lake Nero were explored. CYN and cyr/aoa genes present in environmental samples were examined using HPLC/MS-MS and PCR analysis. A R. raciborskii strain (R104) was isolated and its morphology, toxigenicity and phylogeography were studied. It is supposed that the trigger factor for the strong development of R. raciborskii in Lake Nero in summer 2010 may have been the relatively high water temperature, reaching 29–30 °C. Strain R. raciborskii R104 has straight trichomes and can produce akinetes, making it morphologically similar to European strains. Phylogeographic analysis based on nifH gene and 16S-23S rRNA ITS1 sequences showed that the Russian strain R104 grouped together with R. raciborskii strains isolated from Portugal, France, Germany and Hungary. The Russian strain R104 does not contain cyrA and cyrB genes, meaning that it – like all European strains – cannot produce CYN. Thus, while recent invasion of R. raciborskii into Lake Nero has occurred, morphological, genetic, and toxicological data supported the spreading of this cyanobacterium from other European lakes. Detection of CYN and cyr/aoa genes in environmental samples indicated the cyanobacterium Aphanizomenon gracile as a likely producer of CYN in Lake Nero. The article also discusses data on the global biogeography of R. raciborskii. Genetic similarity between R. raciborskii strains isolated from very remote continents might be related to the ancient origin of the cyanobacterium inhabiting the united continents of Laurasia and Gondwana, rather than comparably recent transoceanic exchange between R. raciborskii populations.

Phosphorus (P) is a vital macronutrient associated with the growth and proliferation of Raphidiopsis raciborskii, an invasive and notorious bloom-forming cyanobacterium. However, the molecular mechanisms involved in P acclimation remain largely unexplored for Raphidiopsis raciborskii. Here, transcriptome sequencing of Raphidiopsis raciborskii was conducted to reveal multifaceted mechanisms involved in mimicking dipotassium phosphate (DIP), β-glycerol phosphate (Gly), 2-aminoethylphosphonic acid (AEP), and P-free conditions (NP). Chlorophyll a fluorescence parameters showed significant differences in the NP and AEP groups compared with the DIP and Gly-groups. Expression levels of genes related to phosphate transportation and uptake, organic P utilization, nitrogen metabolism, urea cycling, carbon fixation, amino acid metabolism, environmental information, the ATP-synthesis process in glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway were remarkably upregulated, while those related to photosynthesis, phycobiliproteins, respiration, oxidative phosphorylation, sulfur metabolism, and genetic information were markedly downregulated in the NP group relative to the DIP group. However, the expression of genes involved in organic P utilization, the urea cycle, and genetic information in the Gly-group, and carbon-phosphorus lyase, genetic information and environmental information in the AEP group were significantly increased compared to the DIP group. Together, these results indicate that Raphidiopsis raciborskii exhibits the evolution of coordination of multiple metabolic pathways and certain key genes to adapt to ambient P changes, which implies that if P is reduced to control Raphidiopsis raciborskii bloom, there is a risk that external nutrients (such as nitrogen, amino acids, and urea) will stimulate the growth or metabolism of Raphidiopsis.

As a successful invasive cyanobacterial species, Raphidiopsis raciborskii is distributed globally and shows a tendency to replace Microcystis aeruginosa in many subtropical and temperate waters, but the ecological traits that contribute to its invasiveness are still unclear. In this study, we found that R. raciborskii occurred in 149 sites in 42 lakes in eastern and central China and coexisted with M. aeruginosa at most sites. Based on field results, a combination of invasion and competition experiments using a biomass gradient to evaluate the invasiveness and competitiveness of R. raciborskii compared with M. aeruginosa was conducted. In invasive groups, both R. raciborskii and M. aeruginosa were shown to have positive specific growth rates, indicating that R. raciborskii could coexist with M. aeruginosa. Furthermore, R. raciborskii was shown to grow faster from invasion while M. aeruginosa reduced growth for invasion. In competitive groups, R. raciborskii reached a higher maximum biomass and grew longer than M. aeruginosa. The specific growth rate of R. raciborskii was not inhibited by M. aeruginosa biomass, whereas the growth of M. aeruginosa was inhibited by R. raciborskii biomass. It was shown during the whole experiment that R. raciborskii tended to replace M. aeruginosa to become dominant owing to its faster growth rate and the eventual decline in growth of M. aeruginosa. With an increase in biomass of M. aeruginosa, the vegetative cell size and filament length of R. raciborskii gradually increased. This study has demonstrated that the inherent invasive traits of R. raciborskii, size differences, niche differences, and relative fitness differences between R. raciborskii and M. aeruginosa are crucial reasons for the invasive success of R. raciborskii. Our results revealed the invasiveness and domination of R. raciborskii from a new perspective.

Blooms of the toxic cyanobacterium, Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii), are becoming a major environmental issue in freshwater ecosystems globally. Our precision prevention and early detection of R. raciborskii blooms rely upon the accuracy and speed of the monitoring method. A duplex digital PCR (dPCR) monitoring approach was developed and validated to detect the abundance and toxin-producing potential of R. raciborskii simultaneously in both laboratory spiked and environmental samples. Results of dPCR were strongly correlated with traditional real time quantitative PCR (qPCR) and microscopy for both laboratory and environmental samples. However, discrepancies between methods were observed when measuring R. raciborskii at low abundance (1 − 105 cells L − 1), with dPCR showing a higher precision compared to qPCR at low cell concentration. Furthermore, the dPCR assay had the highest detection rate for over two hundred environmental samples especially under low abundance conditions, followed by microscopy and qPCR. dPCR assay had the advantages of simple operation, time-saving, high sensitivity and excellent reproducibility. Therefore, dPCR would be a fast and precise monitoring method for the early warning of toxic bloom-forming cyanobacterial species and assessment of water quality risks, which can improve prediction and prevention of the impacts of harmful cyanobacterial bloom events in inland waters.

An early warning scheme is proposed that runs ensembles of inferential models for predicting the cyanobacterial population dynamics and cyanotoxin concentrations in drinking water reservoirs on a diel basis driven by in situ sonde water quality data. When the 10- to 30-day-ahead predicted concentrations of cyanobacteria cells or cyanotoxins exceed pre-defined limit values, an early warning automatically activates an action plan considering in-lake control, e.g. intermittent mixing and ad hoc water treatment in water works, respectively. Case studies of the sub-tropical Lake Wivenhoe (Australia) and the Mediterranean Vaal Reservoir (South Africa) demonstrate that ensembles of inferential models developed by the hybrid evolutionary algorithm HEA are capable of up to 30 days forecasts of cyanobacteria and cyanotoxins using data collected in situ . The resulting models for Dolicospermum circinale displayed validity for up to 10 days ahead, whilst concentrations of Cylindrospermopsis raciborskii and microcystins were successfully predicted up to 30 days ahead. Implementing the proposed scheme for drinking water reservoirs enhances current water quality monitoring practices by solely utilising in situ monitoring data, in addition to cyanobacteria and cyanotoxin measurements. Access to routinely measured cyanotoxin data allows for development of models that predict explicitly cyanotoxin concentrations that avoid to inadvertently model and predict non-toxic cyanobacterial strains.

We tested the hypothesis that a combination of coagulant and ballast could be efficient for removal of positively buoyant harmful cyanobacteria in shallow tropical waterbodies, and will not promote the release of cyanotoxins. This laboratory study examined the efficacy of coagulants [polyaluminium chloride (PAC) and chitosan (made of shrimp shells)] alone, and combined with ballast (lanthanum modified bentonite, red soil or gravel) to remove the natural populations of cyanobacteria collected from a shallow eutrophic urban reservoir with alternating blooms of Cylindrospermopsis and Microcystis. PAC combined with ballast was effective in settling blooms dominated by Microcystis or Cylindrospermopsis. Contrary to our expectation, chitosan combined with ballast was only effective in settling Cylindrospermopsis-dominated blooms at low pH, whereas at pH ≥ 8 no effective flocculation and settling could be evoked. Chitosan also had a detrimental effect on Cylindrospermopsis causing the release of saxitoxins. In contrast, no detrimental effect on Microcystis was observed and all coagulant-ballast treatments were effective in not only settling the Microcystis dominated bloom, but also lowering dissolved microcystin concentrations. Our data show that the best procedure for biomass reduction also depends on the dominant species.

Raphidiopsis raciborskii is a widely distributed, potentially toxic cyanobacterium described as a tropical-subtropical species. However, its occurrence in temperate regions has been expanding. Understanding the environmental factors underlying the expansion and colonization success of Raphidiopsis has been the object of numerous studies. However, less is known regarding its responses to pH and inorganic carbon in water. Thus, the aim of the present study was to investigate the effects of changes in pH and dissolved inorganic carbon on growth and saxitoxins production in the strain R. raciborskii ITEP-A1. We incubated batch cultures with different unbuffered and buffered pH (neutral-acid and alkaline) and inorganic carbon availability (CO2-rich air bubbling and the addition of NaHCO3) to assess the effect of these factors on the growth, toxin production as well as saxitoxins composition of the cyanobacterium. The carbon concentrating mechanism (CCM) system of ITEP-A1 was also characterized by an in silico analysis of its previously sequenced genome. The growth and saxitoxins production of R. raciborskii were affected. The addition of sodium bicarbonate and air bubbling enhanced the growth of the cyanobacterium in alkaline pH. In contrast, saxitoxins production and relative toxicity were decreased. Moreover, significant changes in the cellular composition of saxitoxins were strongly related to pH changes. ITEP-A1 potentially expresses the low-flux bicarbonate transporter BicA, an efficient CCM which uptakes most of its carbon from HCO3−. Hence, increasing the diffusion of CO2 in alkaline eutrophic lakes is likely to increase R. raciborskii dominance, but produce less toxic blooms.

Global increases in atmospheric CO

Due to anthropogenic activities, associated with climate change, many freshwater ecosystems are expected to experience an increase in salinity. This phenomenon is predicted to favor the development and expansion of freshwater cyanobacteria towards brackish waters due to their transfer along the estuarine freshwater-marine continuum. Since freshwater cyanobacteria are known to produce toxins, this represents a serious threat for animal and human health. Saxitoxins (STXs) are classified among the most powerful cyanotoxins. It becomes thus critical to evaluate the capacity of cyanobacteria producing STXs to face variations in salinity and to better understand the physiological consequences of sodium chloride (NaCl) exposure, in particular on their toxicity. Laboratory experiments were conducted on three filamentous cyanobacteria species isolated from brackish (Dolichospermum sp.) and fresh waters (Aphanizomenon gracile and Cylindrospermopsis raciborskii) to determine how salinity variations affect their growth, photosynthetic activity, pigment composition, production of reactive oxygen species (ROS), synthesis of compatible solutes and STXs intracellular quotas. Salinity tolerance was found to be species-specific. Dolichospermum sp. was more resistant to salinity variations than A. gracile and C. raciborskii. NaCl variations reduced growth in all species. In A. gracile, carotenoids content was dose-dependently reduced by NaCl. By contrast, in C. raciborskii and Dolichospermum sp., variations in carotenoids content did not show obvious relationships with NaCl concentration. While in Dolichospermum sp. phycocyanin and phycoerythrin increased within the first 24 h exposure to NaCl, in both A. gracile and C. raciborskii, these pigments decreased proportionally to NaCl concentration. Low changes in salinity did not impact STXs production in A. gracile and C. raciborskii while higher increase in salinity could modify the toxin profile and content of C. raciborskii (intracellular STX decreased while dc-GTX2 increased). In estuaries, A. gracile and C. raciborskii would not be able to survive beyond the oligohaline area (i.e. salinity > 5). Conversely, in part due to its ability to accumulate compatible solutes, Dolichospermum sp. has the potential to face consequent salinity variations and to survive in the polyhaline area (at least up to salinity = 24).

Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii) is a freshwater cyanobacterium potentially producing saxitoxins (STX) and cylindrospermopsin. Its ecophysiological versatility enables it to form blooms in the most diverse types of environments, from tropical to temperate, and from relatively pristine to polluted. In Peri Lake, located in the subtropical south of Brazil, growing populations of STX-producing R. raciborskii have been detected since 1994, posing risks to the use of its waters that supply a population of about 100,000 inhabitants. Despite the existence of a monitoring system for the presence and toxicity of cyanobacteria in Peri Lake water, no assessment has been made in the coastal region, downstream of outflowing lake water, thereby potentially making available a toxic biomass to natural and cultivated shellfish populations in the salt water ecosystem. To address this problem, the present study evaluated environmental variables and STX concentration by profiling the outflowing waters between Peri Lake and the adjacent coastal zone. Laboratory experiments were carried out with three strains of R. raciborskii in order to confirm the effect of salinity on STX production and verify if Perna Perna mussels fed with R. raciborskii cultures would absorb and accumulate STX. Results showed that environmental concentrations of STX reach high levels (up to 6.31 µg L−1 STX eq.), especially in the warmer months, reaching the coastal zone. In laboratory tests, it was found that the strains tolerate salinities between 4 and 6 and that salinity influences the production of STX. In addition, mussels fed with R. raciborskii effectively absorb and accumulate STX, even in typically marine salinities (22 to 30), suggesting that R. raciborskii biomass remains available and toxic despite salinity shock. These results draw attention to the ecological and health risk associated with R. raciborskii blooms, both in the lake environment and in the adjacent marine environment, calling attention to the need to improve the monitoring and management systems for water and shellfish toxicity in the region of interest, as well as other places where toxic cyanobacteria of limnic origin can reach the coastal zone.

The cyanobacterial genera Raphidiopsis and Cylindrospermopsis morphologically resembled each other and are difficult to distinguish, especially when heterocytes do not form in Cylindrospermopsis species. The phylogeny based on multiple gene loci sequences in previous studies cannot divide the strains of these two genera into separate clusters. In the present study, four gene loci 16S rRNA, ITS-L, cpcBA-IGS and rpoC1, sequenced from many Chinese strains (44 Cylindrospermopsis strains and 16 Raphidiopsis strains), were analyzed to infer the phylogenetic relationship between the two genera. According to the 16S rRNA, cpcBA-IGS and rpoC1 gene phylogeny, Cylindrospermopsis and Raphidiopsis strains are intermixed and divide into two or three groups. By contrast, the ITS-L analysis implied that they respectively form two separate and unique clusters. A fragment of 7 bp (RAGAAACT) in ITS-L was conserved for Raphidiopsis strains, which could be distinguished from Cylindrospermopsis strains. A pair of primers targeting the ITS-L fragment specific to Raphidiopsis strains were designed and further verified.

The cyanobacterium Cylindrospermopsis raciborskii is capable of producing toxins including saxitoxin (STX). Few studies have verified the influence of environmental variables on the production of STX and most have only been studied in the laboratory. The goal of this work was to identify the abiotic variables related to STX concentration in situ. The relationship among STX concentration and the physical variables, nutrients and chlorophyll-a (chl-a) concentration was examined in a meso-oligotrophic subtropical coastal lake dominated by C. raciborskii. A generalized linear model was developed, incorporating all variables measured monthly over a 45-month monitoring period. Conductivity and dissolved inorganic nitrogen (DIN) concentration provided the greatest explanatory power for STX concentration in situ. Previous studies suggested that C. raciborskii cells exposed to stress associated with higher ionic concentrations appear to activate the biosynthesis of STX suggesting that STX can elicit changes cell permeability and may contribute to the homeostasis of this organism. An increase of DIN concentration results in a higher concentration of STX which may be related to a reduced metabolic demand, since the uptake of inorganic nitrogen requires less energy than N2-fixation. Thus, increased DIN can favor the growth of C. raciborskii population or improve cellular homeostasis, both potentially increasing STX concentration in the aquatic system, which was observed through a delayed response pattern. The developed model, while providing only a moderate predictive power, can assist in the understanding of the environmental variables associated with increases in STX concentration, and in monitoring and minimizing the risks of toxic blooms.

Predicting algal population dynamics using models informed by experimental data has been used as a strategy to inform the management and control of harmful cyanobacterial blooms. We selected toxic bloom-forming species Microcystis spp. and Raphidiopsis raciborskii (basionym Cylindrospermopsis raciborskii) for further examination as they dominate in 78 % and 17 %, respectively, of freshwater cyanobacterial blooms (cyanoHABs) reported globally over the past 30 years. Field measurements of cyanoHABs are typically based on biomass accumulation, but laboratory experiments typically measure growth rates, which are an important variable in cyanoHAB models. Our objective was to determine the usefulness of laboratory studies of these cyanoHAB growth rates for simulating the species dominance at a global scale. We synthesized growth responses of M. aeruginosa and R. raciborskii from 20 and 16 culture studies, respectively, to predict growth rates as a function of two environmental variables, light and temperature. Predicted growth rates of R. raciborskii exceeded those of M. aeruginosa at temperatures ≳ 25 °C and light intensities ≳ 150 μmol photons m−2 s-1. Field observations of biomass accumulation, however, show that M. aeruginosa dominates over R. raciborskii, irrespective of climatic zones. The mismatch between biomass accumulation measured in the field, and what is predicted from growth rate measured in the laboratory, hinders effective use of culture studies to predict formation of cyanoHABs in the natural environment. The usefulness of growth rates measured may therefore be limited, and field experiments should instead be designed to examine key physiological attributes such as colony formation, buoyancy regulation and photoadaptation. Improving prediction of cyanoHABs in a changing climate requires a more effective integration of field and laboratory approaches, and an explicit consideration of strain-level variability.

Raphidiopsis raciborskii is a tropical toxic cyanobacterium that is rapidly expanding to diverse lake habitats in different climate zones by sophisticated adaptation mechanisms. This meta-analysis investigated correlations of R. raciborskii with water temperature and N:P (nitrogen to phosphorus)-ratios across four lakes with different climates and trophic states by means of long-term time series and the hybrid evolutionary algorithm HEA. The results have shown that in the lakes with temperate and Mediterranean climate, R. raciborskii is strongly correlated with water temperature since germination and growth rely on rising water temperatures in spring. In contrast, there was a weaker correlation with water temperature in subtropical and tropical lakes where pelagic populations of R. raciborskii are overwintering, and are present all year round. However, the highest abundances of R. raciborskii coincided with highest water temperature for the Mediterranean, subtropical and tropical lakes, whilst in the temperate Langer See the highest abundances of R. raciborskii occurred at 24.1 °C, even though temperatures of up to 27 °C were recorded in 2013 and 2014. The correlation of R. raciborskii with N:P-ratios proved to be strongest for the meso- to eutrophic Lake Kinneret (r2 = 0.8) and lowest for the eutrophic Lake Paranoa (r2 = 0.16). However, the assumption has been confirmed that R. raciborskii is growing fastest when waters are N-limited regardless of trophic states. In terms of phenology, the temperate and Mediterranean lakes displayed “fastest growth” in spring and early summer. In contrast, the growing season in subtropical and tropical lakes lasted from spring to autumn most likely because of overwintering populations, and growing importance of direct and indirect biotic regulating factors such as competition, grazing, remineralisation of nutrients along warming climate. In order to carry out a meta-analysis of time series across four different lakes, HEA served as powerful tool resulting in inferential models with predictive capacity for population dynamics of R. raciborskii just driven by water temperature or N:P-ratios, whilst coefficients of determination r2 served as criteria for hypotheses testing.

The cyanobacterium Raphidiopsis raciborskii is a nuisance in freshwater ecosystems. Strains vary in their physiological responses to environmental drivers, thus a greater understanding of the magnitude of strain variation is required to characterize the species. In this study, two strains of R. raciborskii isolated from a tropical Australian water reservoir were grown with and without phosphorus (P) to determine any relative response to P stress. The strains had the same growth rates and under P free conditions, cells grew at the same rate as P replete conditions until day 9 when cell growth ceased. There was no difference in the alkaline phosphatase activity per cell for the P replete and P free conditions, but the level of activity per cell was greater in CS-505 than CS-506. P acquisition genes were identified from the sequenced genomes; both strains contained the same genes, but with differences in copy number of phoA (7 and 6), phnK (3 and 1) and phnH (2 and 1) between CS-505 and CS-506 (respectively). The expression of P acquisition genes under P stress was measured throughout the experiment and shown to vary in magnitude and timing across strains, and in P replete versus P free cultures. In strain CS-505, upregulation of the pstS1 and phoA genes occurred late in the growth phase and into senescence. These genes are involved in phosphate uptake and use of various forms of organic P. For strain CS-506, there was upregulation of the phosphate uptake gene, pit, and organic P utilization genes, phoA, phoU, phnD and phnK, commencing late in the growth phase. Our study shows that despite the fact that these two strains were isolated from the same waterbody, they differed markedly in their gene expression response to P free conditions. This capacity of R. raciborskii to vary in strain responses to P conditions gives the organism flexibility in responding to environmental change, particularly P stress conditions.

Cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii are two harmful species which co-occur and successively dominate in freshwaters globally. Within-species strain variability affects cyanobacterial population responses to environmental conditions, and it is unclear which species/strain would dominate under different environmental conditions. This study applied a Monte Carlo approach to a phytoplankton dynamic growth model to identify how growth variability of multiple strains of these two species affects their competition. Pairwise competition between four M. aeruginosa and eight C. raciborskii strains was simulated using a deterministic model, parameterized with laboratory measurements of growth and light attenuation for all strains, and run at two temperatures and light intensities. 17 000 runs were simulated for each pair using a statistical distribution with Monte Carlo approach. The model results showed that cyanobacterial competition was highly variable, depending on strains present, light and temperature conditions. There was no absolute 'winner' under all conditions as there were always strains predicted to coexist with the dominant strains, which were M. aeruginosa strains at 20°C and C. raciborskii strains at 28°C. The uncertainty in prediction of species competition outcomes was due to the substantial variability of growth responses within and between strains. Overall, this study demonstrates that within-species strain variability has a potentially large effect on cyanobacterial population dynamics, and therefore this variability may substantially reduce confidence in predicting outcomes of phytoplankton competition in deterministic models, that are based on only one set of parameters for each species or strain.

In the study of cyanobacterial communities, molecular methods can be used to detect and quantify toxin-producing strains and assess genotype diversity. These are promising methods for estimating the risk of exposure to the cyanobacterial toxin microcystin (MC), assuming that the abundance of toxic genotypes is related to the MC concentration. This assumption has been tested in many studies, most of which were conducted in temperate and subtropical aquatic environments, and has not always been met. In this study, we collected cyanobacterial samples from a tropical reservoir and tested whether the MC concentration could be explained by variations in the number or ratio of toxin-producing genotypes or by the presence of certain Microcystis genotypes. The genetic diversity of cyanobacteria was determined by cpcBA sequencing, and the proportion of potential MC-producing genotypes was quantified by qPCR using mcyB as the target gene. Cyanobacteria dominated during the entire sampling period (88.4–99.8% of the total phytoplankton biomass), with Microcystis, Anabaena and Cylindrospermopsis occurring as the main genera. Three major Microcystis genotypes were recognized along with other rare or unique genotypes. MCs were detected in all samples (MC-LR, RR and YR), with most concentrations being less than 200 ng L−1. Microcystis was the most abundant group during all the study period and was the main MC-producing genus. Potentially toxic Microcystis cells were present in all samples, varying from 10% to 100% of the total Microcystis. MC concentrations were not correlated with the number or ratio of toxic cells of Microcystis determined by qPCR or with the predominance of certain genotypes. These results indicate the limitations of using molecular methods such as qPCR in MC risk assessment.

Cyanobacteria and their metabolites are recognised as serious water quality issues for water suppliers around the world. Water shortages not only increase the risk of the proliferation of cyanobacteria but also necessitate the development of more effective treatment regimes. Conventional water treatment processes are considered an effective initial barrier in removing cyanobacterial cells intact and without damage. However, the cells can be exposed to a range of pH conditions during these processes. The impact of various pH conditions typical of the water treatment processes was evaluated on common bloom species, Microcystis aeruginosa, Cylindrospermopsis raciborskii and Anabaena circinalis. By using cyanobacterial metabolite analysis and cell viability assays, it was shown that low pH stress (pH below 5), which can occur during coagulation upon the application of metal hydroxide coagulants, occurs quickly and results in considerable cell lysis and metabolite release. It is demonstrated that it is critical to keep the pH of water above 5 (locally) at all times in order to reduce the risk of water contamination with problem metabolites.

Many harmful cyanobacterial genera have strains that can produce potent toxins and other biologically active compounds that present a risk to the health of humans and other animals that consume or contact contaminated water. Cylindrospermopsins (CYNs) are produced by several species of cyanobacteria including Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju. Previous studies have used filtration methods to separate between the particulate and dissolved CYNs pools. Filtration may lyse cells and thus overestimate the dissolved CYNs pool. Here we employed a novel passive sampling technique to measure the proportion of dissolved CYNs in two Australian strains of C. raciborskii over the growth cycle while minimizing potential overestimation of the dissolved CYNs pool. We simultaneously compared the ratios of the two major CYNs produced by Australian strains of C. raciborskii: cylindrospermopsin (CYN) and deoxy-CYN in the particulate and dissolved pools. CYNs stayed within the cells during log phase but accumulated in the water column during stationary and senescent phases. The proportion of deoxy-CYN to CYN differed between strains but increased in both as cells aged. We conclude that while active release or leaking of CYNs from actively growing cells does occur, CYNs in the water column were primarily a result of cell lysis during stationary phase or due to other environmental stressors. The production of CYN and deoxy-CYN were a constitutive process and both the concentration of, ratio between, and release of CYN and deoxy-CYN were strain dependent. Future studies must account for the genetic diversity of CYN producers when investigating the production of CYNs in natural systems.

Microcystis aeruginosa and Cylindrospermopsis raciborskii are two cyanobacterial species that dominate freshwaters globally. Multiple strains of each species with different physiology occur, however, many studies have focused only on one or two strains, limiting our understanding of both strain variation and characterisation of the species. Therefore, in this study we examined the variation in growth and morphology of multiple isolates of both species, isolated from two adjacent Australian reservoirs. Four M. aeruginosa strains (=isolates) (one colony-forming, three single-celled morphology) and eight C. raciborskii isolates (five with straight trichomes, three with coiled trichomes) were cultured individually in a factorial designed experiment with four light intensities (L: 10, 30, 50 and 100μmol photons m-2s-1) and two temperatures (T: 20 and 28°C). The specific growth rate (μ), cell volume, and final cell concentration was measured. The light attenuation coefficient (kj), a measure of self-shading, was calculated. The results showed that the intraspecific variation was greater than the interspecific variation. The μ of all isolates of M. aeruginosa and C. raciborskii ranged from 0.16 to 0.55d-1 and 0.15 to 0.70d-1, respectively. However, at a specific light and temperature the mean μ of all M. aeruginosa isolates and C. raciborskii isolates were similar. At the species level, M. aeruginosa had higher growth rates at higher light intensity but lower temperature (L100T20), while straight C. raciborskii had higher growth rates at lower light intensity but higher temperature (L50T28), and coiled C. raciborskii had higher growth rates at higher light intensity and higher temperature (L100T28). The final cell concentrations of M. aeruginosa were higher than C. raciborskii. However, C. raciborskii isolates had greater variation in μ, kj and cell volume than M. aeruginosa. kj varied with light and temperature, and decreased with surface-to-volume ratio within each species. kj was lower for M. aeruginosa compared to C. raciborskii as expected based on cell size, but interestingly, C. raciborskii coiled isolates had lower kj than the straight isolates suggesting lower effect of self-shading. This study highlights the extent of strain variation to environmental conditions and to species variability.

Cyanobacterial blooms are a current cause for concern globally, with vital water sources experiencing frequent and increasingly toxic blooms in the past decade. These increases are resultant of both anthropogenic and natural factors, with climate change being the central concern. Of the more affected parts of the world, Africa has been considered particularly vulnerable due to its historical predisposition and lag in social economic development. This review collectively assesses the available information on cyanobacterial blooms in Africa as well as any visible trends associated with reported occurrences over the last decade. Of the 54 countries in Africa, only 21 have notable research information in the area of cyanobacterial blooms within the last decade, although there is substantial reason to attribute these blooms as some of the major water quality threats in Africa collectively. The collected information suggests that civil wars, disease outbreaks and inadequate infrastructure are at the core of Africa's delayed advancement. This is even more so in the area of cyanobacteria related research, with 11 out of 21 countries having recorded toxicity and physicochemical parameters related to cyanobacterial blooms. Compared to the rest of the continent, peripheral countries are at the forefront of research related to cyanobacteria, with countries such as Angola having sufficient rainfall, but poor water quality with limited information on bloom occurrences. An assessment of the reported blooms found nitrogen concentrations to be higher in the water column of more toxic blooms, validating recent global studies and indicating that phosphorous is not the only factor to be monitored in bloom mitigation. Blooms occurred at low TN: TP ratios and at temperatures above 12°C. Nitrogen was linked to toxicity and temperature also had a positive effect on bloom occurrence and toxicity. Microcystis was the most ubiquitous of the cyanobacterial strains reported in Africa and the one most frequently toxic. Cylindrospermopsis was reported more in the dry, north and western parts of the continent countries as opposed to the rest of the continent, whilst Anabaena was more frequent on the south eastern regions. In light of the entire continent, the inadequacy in reported blooms and advances in this area of research require critical intervention and action.

Dolichospermum flos-aquae and Cylindrospermopsis raciborskii are two cyanobacteria species which cause harmful blooms around the world. Both these species share the capacity to fix atmospheric nitrogen in heterocytes (cell where fixation occurs). While Dolichospermum can express heterocytes at rather regular intervals across the filament, Cylindrospermopsis can only express heterocytes at the end of the filament. The aim of this study was to experimentally assess the role of heterocyte position in the eco-physiological responses of these bloom forming cyanobacteria. Replicated monocultures of each species were grown at different eutrophication scenarios (limiting and sufficient nitrogen and phosphorus concentrations, in factorial design). Dolichospermum reached high biomass regardless of the nitrogen (and phosphorus) provided, suggesting that this species could bloom in situations with and without nitrogen limitation. In contrast, Cylindrospermopsis reached high biomass only when nitrogen supply was high; its biomass was 15-20 times lower when relying on nitrogen fixation. Hence, despite its ability to fix nitrogen, blooms of Cylindrospermopsis would be expected only under high total nitrogen availability. In Dolichospermum heterocytes occurred only in the scenarios without supplied nitrogen while in Cylindrospermopsis heterocytes occurred regardless of nitrogen availability. Yet, in both species nitrogen fixation occurred (heterocytes were functional) only when nitrogen was limiting, and nitrogen fixation increased significantly at higher phosphorus concentration. Finally, in the absence of supplied nitrogen, filament length in Dolichospermum was the longest, while filaments in Cylindrospermopsis were the shortest (up to 13 times shorter than at nitrogen sufficiency). Therefore, heterocyte expression in Dolichospermum, and filament length in Cylindrospermopsis seem good proxies of nitrogen fixation. The eco-physiological responses recorded here help understand the distribution of these species along nutrient gradients in nature. Fil: Yema, Lilen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Laboratorio de Limnología; Argentina Fil: Litchman, Elena. Michigan State University; Estados Unidos Fil: de Tezanos Pinto, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Laboratorio de Limnología; Argentina

The development of cyanobacterial blooms in inland aquatic ecosystems is greatly promoted by nutrient availability, especially nitrogen and phosphorous. When blooms are dominated by toxigenic species the harmful effects of nutrient loading becomes particularly relevant. Among toxic species, Cylindrospermopsis raciborskii found in South American ecosystems is characterized by the production of saxitoxin and analogs (Paralytic Shellfish Poisoning, PSP), for which the factors that trigger their production have not been elucidated. In this study, the effect of nitrate availability on the relative transcript abundance of two genes (sxtU and sxtI), both involved in different steps of PSP biosynthetic pathway, was addressed in C. raciborskii MVCC19 by qPCR. The relative transcript abundance of both genes significantly increased from the beginning to the end of growth, independent of nitrate availability in the culture medium. Differences between the genes in terms of the levels of relative expression were also found, implying that during growth in nitrate-rich or nitrate-deprived conditions C. raciborskii MVCC19 has the ability to produce different kind of PSP molecules. The presence of nifH transcripts in the nitrogen-depleted treatment confirmed that in the absence of nitrate C. raciborskii fixed atmospheric N2. Moreover, after transferring filaments to nitrate-rich conditions the synthesis of nifH mRNA continued for few hours, suggesting that cell adjustments enabling the utilization of soluble nitrogen sources are not immediate. Our results show that biosynthesis of saxitoxin and analogs in C. raciborskii is not related to nitrate availability, but rather is linked to cyanobacteria growth rate.

Physiological responses to varying dissolved inorganic phosphorus (DIP) levels were examined to explore the acclimation mechanisms of Cylindrospermopsis raciborskii, a nontoxic diazotrophic cyanobacterium. The results showed that the growth rate of C. raciborskii was markedly reduced under phosphorus (P)-deficient conditions. Photosynthetic activity in terms of F-v/F-m (maximum quantum yield), ETRmax (maximum electron transport rate), alpha (photosynthetic efficiency) and I-k (saturation irradiance) was also found to be significantly lower after a 12-day incubation with a Pi concentration of less than 0.05 mg L-1. When supplied with phosphorus concentrations of more than 0.50 mg L-1, C raciborskii showed increased growth rates and reached a relatively stable value, about 0.26 d(-1). Additionally, the content of malodialdehyde (MDA), a lipid peroxidation product reflecting the destruction of lipid membrane by environmental stresses, in Cylindrospermopsis cultured with a low P concentration was 10.81-12.13 times higher than that with a high P concentration. The relative activities of catalase (CAT) and extracellular phosphatase (APA) were found to increase significantly to reduce the stress of P limitation and compensate for P deficiency. These data indicate that C. raciborskii is able to regulate its physiological metabolism in order to acclimate to low ambient DIP environments. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.